Oncogenic tyrosine kinases have proven to be promising targets for the development of highly effective anticancer drugs. However HER family tyrosine kinase inhibitors (TKIs) show only limited activity against HER2-driven cancers despite effective inhibition of EGFR and HER2 in vivo. The reasons for this are unclear. Signaling in trans is a key feature of this multimember family and an abundance of work from our lab and others has shown that TKI sensitivity in these tumors is mediated through the critically important PI3K/Akt pathway, which is driven predominantly through trans-phosphorylation of the kinase-inactive HER3. We have now discovered that although TKIs suppress EGFR and HER2 phosphorylation effectively, phosphorylation of the trans-target HER3 recovers after a transient inhibition and continues to drive PI3K/Akt signaling. This appears to be due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilbrium steady state and is driven largely by redistribution of HER3 to the cell surface. Our overall goal is to understand the molecular mechanisms that underlie HER3 resistance and to re-evaluate the utility of HER TKIs in the treatment of HER2-driven cancers. In aim 1 we propose to confirm the critical role of HER3 in tumor drug resistance by establishing a HER3 siRNA in vivo knock-down model. In addition to this proof-of- principle experiment, we will test several more readily translatable pharmacologic strategies to suppress HER3 signaling and overcome TKI resistance. The transient effects of current TKIs may be clinically relevant, since when used in a pulse dosing schedule preceding chemotherapy, we find that they have a previously unrealized chemosensitization potential. Preliminary data suggests that this chemosensitization is mediated through transient effects on tumor vasculature and possible enhanced tumor delivery of chemotherapeutics. In aim 2 we propose to study this hypothesis using radiologic and histologic studies of tumor vascular perfusion, permeability, and drug delivery. If they are found to support the hypothesis, the radiologic modalities will be translatable to our ongoing clinical studies of pulse chemosensitization. In the third aim, we seek to determine the molecular mechanisms by which HER3 redistributes to the cell surface and overcomes initial suppression by TKIs. Preliminary evidence suggests that Akt-driven negative feedback signaling directly or indirectly regulates HER3 localization. All experimental evidence suggests that certain cancers are driven by HER family oncoproteins, yet HER family TKIs have failed to deliver the promise of this oncogene hypothesis. We believe this is due to the previously unrealized short-lived nature of their effects. Through the proposed research program, we plan to develop treatment strategies to optimally use current HER family TKIs as transient inhibitors, and to develop much more effective therapies that more durably inhibit HER family signaling.